ASYNCHRONOUS ENTRY INTO ANAPHASE INDUCED BY OKADAIC ACID - SPINDLE MICROTUBULE ORGANIZATION AND MICROTUBULE KINETOCHORE ATTACHMENTS

We have found that a brief treatment of either PtK2 cells or stamen ha ir cells of Tradescantia virginiana during metaphase with okadaic acid , a potent protein phosphatase inhibitor, results in asynchronous entr y into anaphase. After this treatment, the interval for the separation of sister chromatids can be expanded from a few seconds to approximat ely 5min. We have performed a series of immunolocalizations of cells w ith anti-tubulin antibodies and CREST serum, asking whether okadaic ac id induces asynchronous entry into anaphase through changes in the org anization of the spindle microtubules or through a loss in the attachm ent of spindle microtubules to the kinetochores. Our experiments clear ly indicate that asynchronous entry into anaphase after phosphatase in hibitor treatment is not the result of either altered spindle microtub ule organization or the long-term loss of microtubule attachment to ki netochores. The kinetochore fiber bundles for all of the separating ch romosomes are normally of uniform length throughout anaphase, but afte r asynchronous entry into anaphase, different groups of kinetochore fi ber bundles have distinctly different lengths. The reason for this dif ference in length is that once split apart, the daughter chromosomes b egin their movement toward the spindle poles, with normal shortening o f the kinetochore fiber bundle microtubules. Thus, okadaic acid treatm ent during metaphase does not affect anaphase chromosome movement once it has begun. Our results suggest that one or more protein phosphatas es appear to play an important role during metaphase in the regulatory cascade that culminates in synchronous sister chromatid separation.